UNIVERSITI PUTRA MALAYSIA IMMUNOREGULATION OF MICROGLIA WITH TOCOTRIENOLS

Transcription

1 UNIVERSITI PUTRA MALAYSIA IMMUNOREGULATION OF MICROGLIA WITH TOCOTRIENOLS TAN SHI WEI FPSK(m)

2 IMMUNOREGULATION OF MICROGLIA WITH TOCOTRIENOLS By TAN SHI WEI Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science FEBRUARY 2012 i

3 Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master of Science IMMUNOREGULATION OF MICROGLIA WITH TOCOTRIENOLS Chairman Faculty : Sharmili Vidyadaran, PhD By TAN SHI WEI FEBRUARY 2012 : Faculty of Medicine and Health Sciences Microglia, the brain macrophages, are the only immune cells in the central nervous system (CNS). Activated microglia are responsible for inflammatory responses and have been noted in the pathophysiology of various neurodegenerative diseases. Continuous activation of microglia resulting in chronic neuroinflammation is thought to exacerbate neuronal damage. Therefore, modulating the inflammatory responses of microglia may be the key to limiting or treating inflammatory events that occur within the CNS parenchyma. Vitamin E is well-known for its antiinflammatory and its anti-oxidative properties. Natural vitamin E consists of eight chemically distinct compounds: -, -, - and -tocopherol; -, -, - and - tocotrienol. Interestingly, tocotrienols has shown better neuroprotective ability than tocopherols. However, to date, there is no approach in studying antiinflammatory effects of tocotrienols on microglia responses. This study was to elucidate the possible regulatory function of tocotrienols on microglia. First, palm -, - and -tocotrienol fragments and Tocomin 50% (a tocopherol/ tocotrienol ii

4 complex) were screened for their ability to reduce nitric oxide (NO) production by BV2 microglia (an immortalized cell line). BV2 cells were treated with tocotrienols at various concentrations (100 nm, 250 nm, 2.5 M, 10 M and 50 M) for 24 hrs and stimulated with 1 g/ml lipopolysaccharide (LPS). Highest concentration of all 4 tocotrienols fragments limited NO production by LPS-stimulated BV2 cells without affecting their cell viability, as determined by the MTS assay. Among the tocotrienols fragments tested, -tocotrienol reduced the NO production most by approximately 50% after 48 hrs of LPS stimulation (p<.05). Hence, -tocotrienol (3.96 g/ml (10 M) and g/ml (50 M)) and Tocomin 50% (47.50 g/ml and g/ml) were chosen for downstream experiments, by investigating their effects on i-nos gene expression, proliferation and CD40 surface marker expression in BV2 cells. -tocotrienol was found not inhibiting i-nos mrna expression to the extent that was expected based on its ability to limit NO production by BV2 cells. Tocomin 50% on the other hand significantly inhibits i-nos gene expression by 51% (p<.05), indicating that the 2 forms of vitamin E have distinct mechanisms for their ability to reduce NO. Utilising the tritiated thymidine proliferation assay, pretreating BV2 cells with -tocotrienol was found to promote proliferation of both resting and LPS-stimulated microglia. On the other hand, when LPS-treated BV2 cells were post-treated with -tocotrienol, cells proliferation rate was found not being affected. Apart from that, with flow cytometric immunophenotyping, both - tocotrienol and Tocomin 50% were discovered to reduce CD40 activation marker expression levels g/ml of Tocomin 50% showed highest reduction on CD40 expression in LPS-stimulated BV2 cells by 32%. The findings from this project suggest a potential role for tocotrienols to limit the inflammatory activities of iii

5 microglia within the CNS and thus may offer a potential therapeutic agent for neurodegenerative and neuroinflammatory diseases. iv

6 Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains IMMUNOREGULASI MIKROGLIA DENGAN TOKOTRIENOL Pengerusi Fakulti : Sharmili Vidyadaran, PhD Oleh TAN SHI WEI FEBRUARI 2012 : Fakulti Perubatan dan Sains Kesihatan Mikroglia, iaitu makrofaj di bahagian otak, adalah satu-satunya sel imun yang terdapat di dalam sistem saraf pusat (CNS). Mikroglia yang aktif memainkan peranan penting dalam tindakbalas keradangan dan ia juga telah dititikberat dalam patofisiologi pelbagai penyakit yang melibatkan kemerosotan neuron. Pengaktifan mikroglia yang berterusan menyebabkan keradangan neuron yang kronik dan dipercayai akan menambah buruk lagi kerosakan neuron. Oleh itu, pengawalan tindakbalas keradangan mikroglia merupakan satu kekunci untuk mengehad atau merawat keradangan yang muncul dalam persekitaran CNS. Sebagai pengetahuan, Vitamin E terkenal dengan ciri-ciri anti-radang dan anti-oksida. Vitamin E yang semulajadi terdiri daripada lapan kompaun yang berlainan secara kimia, iaitu -, -, - dan -tokoferol serta -, -, - dan -tokotrienol. Dengan menakjubnya, tokotrienol telah menunjukkan keupayaan perlindungan neurons yang lebih baik berbanding dengan tokoferol. Walau bagaimanapun, sehingga kini, masih tiada pendekatan dalam kajian tentang kesan-kesan anti-radang tokotrienol terhadap v

7 tindakbalas mikroglia. Kajian ini bertujuan menunjukkan potensi pengawalan tokotrienols terhadap mikroglia. Pertama sekali, fragmen tokotrienol kelapa sawit -, - and -tokotrienol dan Tocomin 50% (kompleks tokoferol/ tokotrienol) telah disaring untuk menguji keupayaan fragmen-fragmen tersebut dalam mengurangkan penghasilan nitrik oksida (NO) oleh BV2 mikroglia (satu imortalisi sel abadi). Sel BV2 telah dirawat menggunakan tokotrienol pada pelbagai kepekatan (100 nm, 250 nm, 2.5 M, 10 M dan 50 M) selama 24 jam dan dirangsang menggunakan 1 g/ml lipopolysaccharide (LPS). Keempat-empat fragmen tokotrienol pada kepekatan tertinggi dapat mengehad penghasilan NO oleh sel BV2 yang dirangsangkan menggunakan LPS tanpa menjejaskan kebolehhidupan sel BV2 tersebut, sebagaimana yang ditentukan oleh ujian MTS. Di antara fragmen-fragmen tokotrienol yang diuji, fragmen -tokotrienol paling banyak mengurangkan penghasilan NO, iaitu sebanyak 50% selepas 48 jam rangsangan LPS (p<.05). Maka, -tokotrienol (3.96 g/ml (10 M) dan g/ml (50 M)) dan Tocomin 50% (47.50 g/ml dan g/ml) telah dipilih untuk eksperimen yang seterusnya, dengan bertujuan menyiasat kesan mereka terhadap ekspresi gen i-nos, proliferasi dan ekspresi penanda permukaan CD40 pada sel BV2. Didapati -tokotrienol tidak merencat ekspresi mrna i-nos seperti yang dijangka berdasarkan keupayaannya dalam mengehad penghasilan NO oleh sel BV2. Manakala, Tocomin 50% dapat merencat ekspresi gen i-nos sel BV2 sebanyak 51% (p<.05), ini menunjukkan kedua-dua jenis vitamin E ini mempunyai mekanisme yang berbeza dalam keupayaan untuk mengurangkan penghasilan NO. Dengan menggunakan asai proliferasi timidina tertritium, didapati sel BV2 yang dipra-rawat menggunakan - tokotrienol menggalakan proliferasi kedua-dua peringkat microglia sama ada dalam vi

8 rehat atau aktif dirangsang oleh LPS. Sebaliknya, apabila mikroglia dirawat dengan -tokotrienol selepas rangsangan LPS, didapati rawatan tersebut tidak memberi kesan kepada kadar pembahagian sel. Di samping itu, dengan teknik sitometri aliran, didapati bahawa kedua-dua -tokotrienol dan Tocomin 50% mempunyai kesan pengurangan taraf ekspresi penanda pengaktifan CD40. Tocomin 50% pada g/ml paling banyak mengurangkan ekspresi CD40 dalam sel BV2 yang dirangsang oleh LPS, iaitu sebanyak 32%. Penemuan daripada projek ini menunjukkan tokotrienol berpotensi dalam mengehad aktiviti keradangan mikroglia dan boleh ditawarkan sebagai satu agen terapeutik untuk merawat penyakit kemerosotan neuron dan keradangan neuron. vii

9 ACKNOWLEDGEMENTS First of all, I would like to take this opportunity to express my deepest and most sincere gratitude to my supervisor, Dr. Sharmili Vidyadaran, for her advice, guidance, encouragement and countless contribution throughout my Master degree. She inspired me all the time and really gave me a push especially when I faced failures. A special note of appreciation also goes to Dr. Maha Abdullah as my co-supervisor for her continual supports and encouragement. My acknowledgement is also extended to all my fellow colleagues in Immunology laboratory including, Pei Shen, Yin Yin, Hema, Zul, Shins, Pooi Pooi, Yip, Rhun Yian, Wai Yee, Malini, Jap Meng, Leslie, Tong, Shin Foong, Chooi Ling, Elizabeth, Choo, Homa, Cin Dee, Steph, Cheryl, Catherine, Chou Min, Cini, Kak Aisyah, Mr. Anthony, Marshitah and Kak Zura who have made the last two years an enjoyable experience for me. My heartfelt thanks to my family members especially my parents for their love, dedications and endless moral support. I also appreciate the motivation and support from my understanding housemates and friends to proceed for completing my degree. Next, I would like to thank the Carotech Sdn. Bhd. (Malaysia) and Assoc. Prof. Dr. Thameem Dheen from National University Singapore (NUS) for providing me the main ingredient : tocotrienols and BV2 microglia cell line, respectively for my study. Last but not least, I would also like to thank UPM Graduate Research Fellowship Scheme (GRF) for my personal financial support. viii

10 I certify that a Thesis Examination Committee has met on 15 February 2012 to conduct the final examination of Tan Shi Wei on her thesis entitled Immunoregulation of Microglia with Tocotrienols in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March The Committee recommends that the student be awarded the Master of Science. Members of Thesis Examination Committee were as follows: Rozita binti Rosli, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Daud Ahmad bin Israf Ali, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Huzwah binti Khazaai, PhD Senior Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Durriyyah Sharifah Hasan Adli, PhD Associate Professor Faculty of Science Universiti Malaya (External Examiner) SEOW HENG FONG, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 28 June 2012 ix

11 This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Sharmili Vidyadaran, PhD Senior Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Maha Abdullah, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member) BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: x

12 DECLARATION I declare that the thesis is my original work except for the quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution. TAN SHI WEI Date: 15 February 2012 xi

13 TABLE OF CONTENTS Page ABSTRACT ABSTRAK ACKNOWLEDGEMENTS DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATION CHAPTER ii v viii xi xv xvi xviii 1 INTRODUCTION 1 Rationale of the Study 4 Objectives of the Research 5 2 LITERATURE REVIEW Neuroinflammation Microglia Short History of Microglia Origin and Cell Lineage of Microglia Microglia Possess Great Heterogeneity in Distribution and Phenotype Resting Microglia Activated Microglia and Their Associated Molecules Lipopolysaccharide as an Inducer for Microglia Activation Role of Microglia in Neurodegenerative Diseases Microglia in Diseases: Neuroprotective or Neurotoxic? Incidence of Microglia in Neurodegenerative Diseases Treatment Approaches for Neurodegenerative Diseases Natural Vitamin E Family: Tocopherols and Tocotrienols Tocotrienols Structure of Tocotrienols Natural Sources of Tocotrienols Bioavailability of Tocotrienols The Role of -Tocopherol Transfer Protein (TTP) in Tocotrienols Bioavailability Distribution of Tocotrienols in Different Tissues Natural Neuroprotective Agent: Tocotrienols Tocotrienols are more neuroprotective than Tocopherols Bioavailability of Tocotrienols in Central Nervous System 46 3 MATERIALS AND METHODS 48 xii

14 3.1 BV2 Cell Culture Cell Culture Activation Vitamin E Treatment Griess Assay Principle Cell Seeding and Activation Griess Reaction Establishment of NO Standard Curve Determination of NO Production MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4- sulfophenyl)-2h-tetrazolium] Assay Principle Methodology Tritiated Thymidine Incorporation As say Principle Methodology Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Principle RNA Isolation Reverse-Transcription Polymerase Chain Reaction (PCR) Immunophenotyping Principle Methodology Statistical Analysis 62 4 RESULTS Characterisation of BV2 Cell Growth Screening Tocotrienol Fragments for Nitric Oxide-Reducing Capacity Determined Using the Griess Assay Determination of Ideal LPS Concentration Effects of Tocotrienols Fragments and Tocomin 50% on NO Production of BV2 Cells Effects of Tocotrienols Fragments and Tocomin 50% on Cell Viability of BV2 Cells Immunomodulatory Effects of -Tocotrienol and Tocomin 50% on BV2 Cells Determination of NO Production in -Tocotrienol and Tocomin 50% Treated BV2 Cells Effects of -Tocotrienol and Tocomin 50% on Viability of BV2 Cells Determined with the MTS Assay 80 xiii

15 4.3.3 Effects of -Tocotrienol and Tocomin 50% on BV2 Cell Proliferation Determined Using the Tritiated Thymidine Incorporation Assay Effects of -Tocotrienols and Tocomin 50% on CD40 Expression of BV2 Cells 86 5 DISCUSSION BV2 Cultures Immunomodulatory Effects of Tocotrienols on Microglia Inhibitory Effects of Tocotrienols on NO Production of BV2 cells without Affecting Cell Viability Tocotrienols Promote Proliferation of Both Resting and LPSstimulated Microglia Tocotrienol and Tocomin 50% Inhibit CD40 expression of Both Resting and LPS-stimulated Microglia CONCLUSIONS AND FURTHER RECOMMENDATIONS Conclusion Recommendations for Future Studies 112 REFERENCES 114 APPENDIX I - SOLUTIONS AND REAGENTS 132 APPENDIX II- DATA SETS 146 APPENDIX III-PHAGOCYTIC ACTIVITY OF BV2 MICROGLIA 164 LIST OF PUBLICATIONS 170 BIODATA OF THE STUDENT 174 xiv